CN115317441A - Microneedle patch loaded with dacarbazine and manganese salt, preparation method thereof and application thereof in melanoma treatment - Google Patents
Microneedle patch loaded with dacarbazine and manganese salt, preparation method thereof and application thereof in melanoma treatment Download PDFInfo
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- CN115317441A CN115317441A CN202211152607.2A CN202211152607A CN115317441A CN 115317441 A CN115317441 A CN 115317441A CN 202211152607 A CN202211152607 A CN 202211152607A CN 115317441 A CN115317441 A CN 115317441A
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- dacarbazine
- manganese salt
- microneedle patch
- manganese
- degradable polymer
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/32—Manganese; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a micro-needle patch loaded with dacarbazine and manganese salt, a preparation method thereof and application thereof in melanoma treatment. The drug-loaded micro-needle can effectively inhibit the growth and metastasis of tumors, effectively prevent the recurrence of the tumors, and realize the efficient killing of the melanoma.
Description
Technical Field
The invention belongs to the technical field of medical appliance preparation, and particularly relates to a micro-needle patch loaded with dacarbazine and manganese salt, a preparation method of the micro-needle patch and application of the micro-needle patch in melanoma treatment.
Background
Melanoma (melaoma) is caused by abnormal melanocyte hyperproliferation and is frequently found in malignant tumors of the skin, and according to statistics, about 20 ten thousand new cases of Melanoma exist in the world every year, and nearly 7 ten thousand people die of Melanoma and related diseases. The annual growth rate of the disease rate is 3% -5%, and the cancer is one of malignant tumors with the fastest global disease rate and is the second fatal cancer after the leukemia. Currently, primary focus surgical excision or radiotherapy is mainly adopted clinically for early melanoma, the melanoma is often rapid in disease development, relapses and has extremely high metastasis rate, and malignant melanoma cells can have skin transitional lesion and metastasis to lymph nodes or metastasize to organs such as lung, liver, spleen, brain and the like along with blood. Melanoma has high immunogenicity, and abundant immune cells exist around tumor cells. Immunotherapy is not affected by the condition of tumor mutation, and the therapeutic effect is mainly dependent on the immune state of the patient. Therefore, immunotherapy is probably the most promising therapeutic approach for the treatment of malignant melanoma. Recent cancer immunotherapy strategies have been developed based on the modulation of adaptive immune responses. Although the improvement of adaptive immune response has some anticancer effect, it still cannot meet the clinical requirement of cancer treatment, and some auxiliary immune regulation means are needed. Given that innate immunity is the first barrier to host cell defense, and is the bridge to adaptive immunity, activating innate immunity has become an emerging strategy to accelerate tumor immunotherapy.
In the tumor response, dayThe cGAS-STING signaling pathway of the immune system can then activate Dendritic Cells (DCs), tumor-specific CD8 + T cells and natural killer cells (NK), thereby initiating systemic anti-tumor immunity and thereby killing cancer cells. In recent years, studies have shown that Mn is present 2+ Can be used as an agonist to effectively promote the activation of cGAS and STING. Immunology (Immunity, 2018, vol.48, p.675-687) reported Mn 2+ Plays a key role in host defense against DNA viruses. Mn (Mn) 2+ Released from membrane-enclosed organs after viral infection, accumulated in the cytoplasm, and bound directly to cGAS. The sensitivity of the cGAS to double-stranded DNA (dsDNA) and the enzymatic activity thereof is enhanced, so that the cGAS can generate cGAMP in the presence of low-concentration dsDNA, thereby activating STING, and leading the body to generate type I IFN and the antiviral capacity of a host. As the Research proceeds, mn was reported in Cell Research (Cell Research,2020, vol.30, p.966-979) 2+ cGAS can also be activated directly (totally independent of dsDNA), and Mn 2+ Catalysis with Mn 2+ The process by which dsDNA catalyzes cGAS to synthesize cGAMP is significantly different. Mn 2+ cGAS binding leads to a similar change in cGAS conformation as DNA-cGAS binding, but the structure of the catalytic center is significantly different, leading to a large change in cGAMP synthesis at the cGAS catalytic center. This study further demonstrates Mn 2+ Is the second cGAS activator outside of DNA. Thus, mn 2+ Has great potential in activating cGAS-STING signaling pathway to generate anti-tumor immune response. In addition, dacarbazine, as an alkylating agent, can cause DNA damage to tumor cells, which is in turn associated with Mn 2+ The mediated cGAS-STING pathway has a coordinating effect and can strengthen the effect. At present, the dacarbazine is mainly used in clinic and is citric acid dacarbazine for injection, the dacarbazine needs to be prepared for use, and the solution turns into light red after being placed for a long time.
Disclosure of Invention
The invention provides a micro-needle patch loaded with dacarbazine and manganese salt, a preparation method thereof and application thereof in melanoma treatment, and aims to solve the technical problems that: construction of transdermal drug delivery systemsMn in the present 2+ And the dacarbazine is accurately released, the toxic and side effects are reduced, the tumor microenvironment is improved, and a cGAS-STING signal pathway is activated to realize the immunotherapy of the tumor.
In order to solve the technical problem, the invention adopts the following technical scheme:
the invention firstly discloses a micro-needle patch loaded with dacarbazine and manganese salt, which is characterized in that: the microneedle patch comprises a back weighing layer and a needle body array arranged on the back weighing layer; the back weighing layer and the needle body array are formed by molding a water solution of a degradable polymer, and the dacarbazine and manganese salt are loaded in the water solution of the degradable polymer for molding the needle body array.
Further, the manganese salt is at least one of manganese sulfate, manganese chloride, manganese nitrate and manganese acetate.
Further, in the aqueous solution of the degradable polymer for forming the backing layer, the concentration of the degradable polymer is 1-2g/mL.
Further, in the aqueous solution of the degradable polymer for molding into the needle array, the concentration of the degradable polymer is 0.1-0.2g/mL, and the mass ratio of the manganese salt to the degradable polymer is 1:2 to 10, the mass ratio of the dacarbazine to the manganese salt is 0.5 to 1:1
Further, the degradable polymers used by the backing layer and the needle body array are at least one selected from hyaluronic acid, gelatin, polyvinylpyrrolidone and polyvinyl alcohol.
Furthermore, each needle body in the needle body array is in a conical shape with the height of 400-1000 mu m and the bottom end diameter of 150-400 mu m.
The invention also discloses a preparation method of the microneedle patch loaded with the dacarbazine and the manganese salt, which comprises the following steps:
dissolving a degradable polymer in deionized water to obtain a back weighing layer solution;
dissolving the degradable polymer in deionized water, adding dacarbazine and manganese salt, and stirring at room temperature in a dark place for 12-24 hours to obtain a needle body mixed solution;
filling the needle body mixed solution into the needle body micropores of the microneedle mould by a micro-transfer molding method, removing redundant parts except the micropores, adding a back layer solution above the micropores, drying and demoulding to obtain the microneedle patch carrying the dacarbazine and the manganese salt.
The micro-needle patch loaded with the dacarbazine and the manganese salt, which is obtained by the invention, can be used for treating melanoma through transdermal administration and inhibiting metastasis. The invention constructs a bioresponse polymer microneedle drug release system loaded with manganese salt and a chemotherapeutic drug (dacarbazine) for activating innate immunity and adaptive immunity to treat melanoma. Released Mn 2+ And the chemotherapeutic drug dacarbazine induces killing of melanoma cells by ICD and initiates exposure to tumor-specific antigenic molecules including CRT, ATP, HMGB1, IFN- β, CXCL10, etc., thereby activating adaptive immunity; mn 2+ The activation of cGAS-STING-I type interferon can be strengthened during the accumulation of tumor cells, innate immunity is activated, the secretion of IFN-beta is increased, and finally, good tumor inhibition effect of primary and metastatic tumors is realized, so that an experimental basis and data support are provided for improving the effective response rate of the current clinical tumor immunotherapy.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention prepares the soluble microneedle loaded with the dacarbazine and the manganese salt for the first time, has simple preparation method, mild reaction conditions, uniform appearance of the obtained product and lower production cost, and is suitable for industrial large-scale production.
2. The microneedle loaded with the dacarbazine and the manganese salt, prepared by the invention, can realize quick dissolution, realizes more accurate drug release by transdermal drug delivery on the upper epidermis of a tumor part, and has smaller dosage and better biocompatibility.
3. The microneedle prepared by the invention can release Mn 2+ Thereby consuming the overexpressed GSH, mn in the tumor 2+ Can also react with endogenous H through Fenton-like reaction 2 O 2 OH is generated, so that the redox homeostasis of a tumor microenvironment is changed, and the tumor cell killing is realized.
4. The microneedle prepared by the invention can release Mn 2+ Activation of cGAS-STING signalingThe preparation is expected to promote immune cells to generate I-type IFN and related antitumor cytokines, so that infiltration of cytotoxic T cells and NK cells at tumor parts is increased, and effective immunotherapy of melanoma is realized.
5. The invention can realize long-time storage at room temperature by combining the dacarbazine into the micro-needles to form a solid, and the dacarbazine is released locally through the micro-needles in a transdermal manner, so that the dosage and the toxic and side effects of the dacarbazine are obviously reduced, a stronger immune effect can be caused, and a better curative effect is reflected.
Drawings
Fig. 1 is an optical picture of the microneedle prepared in example 1;
fig. 2 is an inverted fluorescence microscope image of the microneedle prepared in example 1 in a bright field;
fig. 3 is a Scanning Electron Microscope (SEM) photograph of the microneedle prepared in example 1;
fig. 4 is a graph showing uv absorption of the microneedles prepared in example 2 dissolved in phosphate buffered saline;
FIG. 5 is a graph of the volumetric changes of the dissected tumor from each treatment group of example 6;
FIG. 6 is a graph of HE staining of dissected tumor tissue from each treatment group of example 6;
FIG. 7 is a TUNEL staining of dissected tumor tissue from each treatment group of example 6;
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
This example prepares microneedles without dacarbazine and manganese salts:
1300mg of hyaluronic acid is weighed and added into 10mL of deionized water and stirred for 24 hours in the dark to form a transparent uniform colloidal mixture. It was transferred to a 10mL beaker and the microneedle mould was added thereto. The beaker was then placed in a centrifuge tube and centrifuged at 4000 rpm for 5min.
And after the centrifugation is finished, taking out the mold in the beaker, removing the part except the micropores, adding 1.5g/mL PVP solution, then putting the mold in an oven at 40 ℃ for drying overnight in the dark, and finally slightly stripping the formed microneedle from the mold.
Fig. 1 is an optical image of the microneedle prepared in this example, and it can be seen from the image that the back symmetric layer of the microneedle patch is a square with a side length of 9 mm.
Fig. 2 is a bright field side view of an inverted fluorescence microscope (Olympus CKX41 biological inverted microscope) of a microneedle prepared according to this example, from which the sharp needle portion of the microneedle can be seen.
Fig. 3 is a scanning electron microscope image (ZEISS GeminiSEM 300 field emission scanning electron microscope) of the microneedle prepared in this example, and the overall appearance of the microneedle can be clearly seen.
Example 2
This example prepares microneedles containing dacarbazine only:
325mg of dacarbazine (the dacarbazine accounts for 25% of the mass of the hyaluronic acid) and 1300mg of hyaluronic acid are weighed and added into 10mL of deionized water to be stirred for 24 hours in a dark place to form a milky uniform colloidal mixture. It was transferred to a 10mL beaker and a microneedle mould (same as in example 1) was added thereto. The beaker was then placed in a centrifuge tube and centrifuged at 4000 rpm for 5min.
And after the centrifugation is finished, taking out the mold in the beaker, removing the part except the micropores, adding 1.5g/mL PVP solution, then putting the mold in an oven at 40 ℃ for drying overnight in the dark, and finally slightly stripping the formed microneedle from the mold.
The microneedles were dissolved in 8mL of phosphate buffered saline and the uv absorption of dacarbazine was measured by uv spectrophotometer for convenient quantification, with a maximum absorption peak at 328nm as shown in figure 4.
Example 3
This example prepares microneedles containing only manganese salt:
650mg of manganese chloride and 1300mg of hyaluronic acid are weighed into 10mL of deionized water and stirred for 24 hours in the dark to obtain a brown uniform colloidal mixture. It was transferred to a 10mL beaker and a microneedle mould (same as in example 1) was added thereto. The beaker was then placed in a centrifuge tube and centrifuged at 4000 rpm for 5min.
And after the centrifugation is finished, taking out the mold in the beaker, removing the part except the micropores, adding 1.5g/mL PVP solution, then putting the mold in an oven at 40 ℃ for drying overnight in the dark, and finally slightly stripping the formed microneedle from the mold.
When the microneedle in this embodiment is placed in a potassium permanganate solution, a color development change is caused because a neutralization reaction occurs between the divalent manganese ions in the microneedle and the heptavalent manganese ions in the potassium permanganate to form manganese ions of an intermediate valence state, thereby causing a color change.
Example 4
In this example, the mass ratio of dacarbazine to manganese salt is 0.5:1 preparing a microneedle:
325mg of dacarbazine, 650mg of manganese chloride and 1300mg of hyaluronic acid are weighed into 10mL of deionized water and stirred for 24h in the dark to form a milky homogeneous colloidal mixture. It was transferred to a 10mL beaker and a microneedle mould (same as in example 1) was added thereto. The beaker was then placed in a centrifuge tube and centrifuged at 4000 rpm for 5min.
And after the centrifugation is finished, taking out the mold in the beaker, removing the part except the micropores, adding 1.5g/mL PVP solution, then putting the mold in an oven at 40 ℃ for drying overnight in the dark, and finally slightly stripping the formed microneedle from the mold.
Example 5
In this embodiment, the mass ratio of dacarbazine to manganese salt is 1:1 preparing a microneedle:
650mg of dacarbazine, 650mg of manganese chloride and 1300mg of hyaluronic acid are weighed and added into 10mL of deionized water, and stirred for 24 hours in the dark to form a milky uniform colloidal mixture. It was transferred to a 10mL beaker and a microneedle mould (same as in example 1) was added thereto. The beaker was then placed in a centrifuge tube and centrifuged at 4000 rpm for 5min.
And after the centrifugation is finished, taking out the mold in the beaker, removing the part except the micropores, adding 1.5g/mL PVP solution, then putting the mold into an oven at 40 ℃ for drying overnight in a dark place, and finally slightly stripping the formed microneedle from the mold.
Example 6
This example tests the therapeutic effect of the microneedles prepared in the above examples on melanoma according to the following steps:
mice were randomly grouped: control microneedle group, dacarbazine microneedle group, manganese salt microneedle group, dacarbazine and manganese salt 0.5: group 1 and dacarbazine with manganese salt 1:1 group of 5 mice each. The modeling method is to inject mouse melanoma cells (B16F 10) into the back to construct a B16F10 subcutaneous tumor implantation model. The treatment method is that when the tumor volume is about 100mm 3 Microneedle patches were attached to the tumor sites of the mice and treated according to groups once every three days.
Control microneedle group: administering the pure hyaluronic acid microneedle of example 1 to the molded mouse;
dacarbazine microneedle set: administering the dacarbazine-only microneedles of example 2 to the molded mice;
manganese salt micro-needle group: administering the manganese salt-only microneedles of example 3 to the molded mice;
0.5: group 1: the molded mice were administered with the dacarbazine and manganese salts of example 4 at a mass ratio of 0.5: 1;
1: group 1: the mass ratio of dacarbazine to manganese salt of example 5 was 1:1.
The volume of the mouse tumor was measured daily and calculated and recorded according to the calculation formula of volume = a × b/2 (a is the maximum length of the mouse tumor and b is the minimum length of the mouse tumor). And (3) killing the mice and taking out the tumors until 14 days are finished, carrying out immunohistochemistry and immunofluorescence pathological analysis on tumor tissues, detecting corresponding necrosis and apoptosis factors, and verifying the killing effect of the microneedles carrying the dacarbazine and the manganese salt on the tumors in the animal bodies.
Fig. 5 is a graph showing the volume change of tumor tissue dissected from each treatment group in this example. As can be seen from the figure, the ratio of dacarbazine to manganese salt 1: the tumor size of group 1 was much smaller than that of the other groups after treatment.
FIG. 6 is a graph of HE staining of dissected tumor tissue from each treatment group in this example. As can be seen from the figure, blue nuclei were more abundant and intact in the control microneedle group and the manganese salt microneedle group, indicating less tumor cell necrosis. The micro-needle group carrying dacarbazine has necrosis in different degrees, which shows that the dacarbazine and the manganese salt have certain killing effect on tumor cells. And the dacarbazine and manganese salt group has the strongest killing effect, the number of blue cell nucleuses is small and the blue cell nucleuses are broken, which shows that the prepared micro-needle carrying the dacarbazine and the manganese salt can effectively cause tumor cell necrosis, and proves that the micro-needle has an obvious treatment effect on melanoma.
FIG. 7 is a TUNEL staining of dissected tumor tissue from each treatment group of this example. As can be seen from the figure, compared with the control micro-needle group and the manganese salt micro-needle group, which represent the absence of green fluorescence of apoptosis, the dacarbazine micro-needle group and the dacarbazine and manganese salt micro-needles have different degrees of apoptosis, which shows that the dacarbazine and the manganese salt have certain killing effect on tumor cells. And the dacarbazine and manganese salt group has the strongest killing effect, so that the green fluorescence is generated most, which shows that the prepared micro-needle carrying the dacarbazine and the manganese salt can effectively cause the tumor cell apoptosis, and proves that the micro-needle has obvious treatment effect on the melanoma.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A microneedle patch loaded with dacarbazine and a manganese salt is characterized in that: the microneedle patch comprises a back weighing layer and a needle body array arranged on the back weighing layer; the back weighing layer and the needle body array are formed by molding a water solution of a degradable polymer, and the dacarbazine and manganese salt are loaded in the water solution of the degradable polymer for molding the needle body array.
2. The microneedle patch carrying dacarbazine and a manganese salt as claimed in claim 1, wherein: the manganese salt is at least one of manganese sulfate, manganese chloride, manganese nitrate and manganese acetate.
3. The microneedle patch carrying dacarbazine and a manganese salt as claimed in claim 1, wherein: in the aqueous solution of the degradable polymer for forming the backing layer, the concentration of the degradable polymer is 1-2g/mL.
4. The microneedle patch carrying dacarbazine and a manganese salt as claimed in claim 1, wherein: in the aqueous solution of the degradable polymer for molding the needle array, the concentration of the degradable polymer is 0.1-0.2g/mL, and the mass ratio of the manganese salt to the degradable polymer is 1:2 to 10, the mass ratio of the dacarbazine to the manganese salt is 0.5 to 1:1.
5. a microneedle patch carrying dacarbazine and a manganese salt as claimed in claim 1, wherein: the back weighing layer and the degradable polymer used by the needle body array are respectively and independently selected from at least one of hyaluronic acid, gelatin, polyvinylpyrrolidone and polyvinyl alcohol.
6. The microneedle patch carrying dacarbazine and a manganese salt as claimed in claim 1, wherein: each needle body in the needle body array is in a conical shape with the height of 400-1000 mu m and the bottom end diameter of 150-400 mu m.
7. A method for preparing a microneedle patch loaded with dacarbazine and a manganese salt according to any one of claims 1 to 6, comprising the steps of:
dissolving a degradable polymer in deionized water to obtain a back weighing layer solution;
dissolving the degradable polymer in deionized water, adding dacarbazine and manganese salt, and stirring at room temperature in a dark place for 12-24h to obtain a needle body mixed solution;
filling the needle body mixed solution into the needle body micropores of the microneedle mould by a micro-transfer molding method, removing redundant parts except the micropores, adding a back layer solution above the micropores, drying and demoulding to obtain the microneedle patch carrying the dacarbazine and the manganese salt.
8. Use of the microneedle patch carrying dacarbazine and a manganese salt according to any one of claims 1-6 for the preparation of a medicament for the treatment of melanoma and for inhibiting metastasis.
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Cited By (1)
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| CN115804842A (en) * | 2023-01-06 | 2023-03-17 | 潍坊医学院 | Dacarbazine nano microneedle preparation as well as preparation method and application thereof |
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| CN108785244A (en) * | 2018-06-20 | 2018-11-13 | 华中科技大学 | A kind of soluble micropin and preparation method thereof supporting dewatering medicament |
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